I. Methods for making novel binding partners
Binding partners are substances that specifically bind to one another, usually through noncovalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
Binding partners have been produced in the past by a variety of methods including; harvesting them from nature (e.g., antibody-antigen, and ligand-receptor pairings) and by adventitious identification (e.g. traditional drug development employing random screening of candidate molecules). In some instances these two approaches have been combined. For example, variants of proteins or polypeptides, such as polypeptide fragments, have been made that contain key functional residues that participate in binding. These polypeptide fragments, in turn, have been derivatized by methods akin to traditional drug development. An example of such derivatization would include strategies such as cyclization to conformationally constrain a polypeptide fragment to produce a novel candidate binding partner.
The problem with prior art methods is that naturally occurring ligands may not have proper characteristics for all therapeutic applications. Furthermore, methods for making non-naturally occurring synthetic binding partners are often expensive and difficult, usually requiring complex synthetic methods to produce each candidate. The inability to characterize the structure of the resulting candidate so that rational drug design methods can be applied for further optimization of candidate molecules further hampers these methods.
In an attempt to overcome these problems, Geysen (Geysen, Immun. Today, 6:364-369 [1985]); and (Geysen et al., Mol. Immun., 23:709-715 [1986]) has proposed the use of polypeptide synthesis to provide a framework for systematic iterative binding partner identification and preparation. According to Geysen et al., Ibid, short polypeptides, such as dipeptides, are first screened for the ability to bind to a target molecule. The most active dipeptides are then selected for an additional round of testing comprising linking, to the starting dipeptide, an additional residue (or by internally modifying the components of the original starting dipeptide) and then screening this set of candidates for the desired activity. This process is reiterated until the binding partner having the desired properties is identified.
The Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure. As rounds of iterative selection progress, random interactions accelerate among the various substituent groups of the polypeptide so that a true random population of interactive molecules having reproducible higher order structure becomes less and less attainable. For example, interactions between side chains of amino acids, which are sequentially widely separated but which are spatially neighbors, freely occur. Furthermore, sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule. Such complex interactions are facilitated by the flexibility of the polyamide backbone of the polypeptide candidates. Additionally, candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.
To overcome many of the problems inherent in the Geysen approach, biological selection and screening has been chosen as an alternative approach. Biological selections and screens are powerful tools to probe protein function and to isolate variant proteins with desirable properties (Shortle, Protein Engineering, Oxender and Fox, eds., A. R. Liss, Inc., New York, pp. 103-108 [1988]) and Bowie et al., Science, 247:1306-1310 [1990]). However, a given selection or screen is applicable to only one or a small number of related proteins.
Recently, Smith and coworkers (Smith, Science, 228: 1315-1317 [1985]) and Parmley and Smith, Gene, 73:305-318 [1985] have demonstrated that small protein fragments (10-50 amino acids) can be "displayed" efficiently on the surface of filamentous phage by inserting short gene fragments into gene III of the fd phage ("fusion phage"). The gene III minor coat protein (present in about 5 copies at one end of the virion) is important for proper phage assembly and for infection by attachment to the pili of E. coli (see Rasched et al., Microbiol. Rev., 50: 401-427 [1986]). Recently, "fusion phage" have been shown to be useful for displaying short mutated peptide sequences for identifying peptides that may react with antibodies (Scott et al., Science 249: 386-390, [1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A. 87:6378-6382, [1990]). or a foreign protein (Devlin et al., Science, 249: 404-406 [1990]).
Ladner (WO 90/02802) discloses a method for selecting novel binding proteins displayed on the outer surface of cells and viral particles where it is contemplated that the heterologus proteins may have up to 164 amino acid residues. The method contemplates isolating and amplifying the displayed proteins to engineer a new family of binding proteins having desired affinity for a target molecule. More specifically, Ladner describes a "fusion phage" displaying proteins having "initial protein binding domains" ranging from 46 residues (crambin) to 164 residues (T4 lysozyme) fused to the M13 gene III coat protein. Small fusion proteins, such as BPTI, are preferred when the target is a protein or macromolecule, while larger fusion proteins, such as T4 lysozyme, are preferred for small target molecules such as steroids because such large proteins have clefts and grooves into which small molecules can fit. The preferred protein, BPTI, is proposed to be fused to gene III at the site disclosed by Smith et al. or de la Cruz et al., J. Biol. Chem., 263: 4318-4322 [1988], or to one of the terminii, along with a second synthetic copy of gene III so that "some" unaltered gene III protein will be present. Ladner does not address the problem of successfully panning high affinity peptides from the random peptide library which plagues the biological selection and screening methods of the prior art.
To overcome problems associated with the Ladner approach, Bass, S., et al., Proteins 8:309-314 (1990) have devised a method for displaying single copies of mutant fusion proteins, especially human growth hormone, on the surface of the filamentous phage M13. This expression system allows large proteins with discontinuous binding epitopes to be displayed on the surface of the filamentous phage and permits biological selections to be applied to mutant gene III fusions. These authors do not describe bivariant biological selection of fusion proteins displayed on mammalian cell surfaces.
II. Tissue-type Plasminogen Activator (t-PA) variants
A substantially pure form of t-PA was first produced from a natural source and tested for in vivo activity by Collen et al., U.S. Pat. No. 4,752,603 issued Jun. 21, 1988 (see also Rijken et al., J. Biol. Chem., 256:7035 [1981]). Pennica et al. (Nature, 301:214 [1983]) determined the DNA sequence of t-PA and deduced the amino acid sequence from this DNA sequence (see U.S. Pat. No. 4,766,075 issued Aug. 23, 1988).
Research on the structure of t-PA has identified the molecule as having five domains. Each domain has been defined with reference to homologous structural or functional regions in other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of t-PA, as the finger (F) domain from amino acids 1 to about 44, the growth factor (G) domain from about amino acids 45 to 91 (based on homology with EGF), the kringle-1 (K1) domain from about amino acids 92-173, the kringle-2 (K2) domain from about amino acids 180 to 261, and the serine protease (P) domain from about amino acid 264 to the carboxyl terminus at amino acid 527. These domains are situated essentially adjacent to each other, and some are connected by short "linker" regions. These linker regions bring the total number of amino acids in the mature polypeptide to 527.
Each domain is believed to confer certain biologically significant properties on the t-PA molecule. The finger domain is thought to be important in the high binding affinity of t-PA to fibrin. Structural determinants for plasma clearance are thought to be on the finger, growth factor, and kringle-1 domains. The kringle-2 domain is responsible for binding to lysine. The serine protease domain is responsible for the enzymatic activity of t-PA and the fibrin specificity.
t-PA variants with decreased clearance have been prepared by deleting individual amino acids, partial domains, or complete domains from the molecule. For example, removal of part or all of the finger domain of t-PA as described in U.S. Pat. No. 4,935,237 (issued Jun. 19, 1990) results in a molecule with decreased clearance, although it has substantially diminished fibrin-binding characteristics. Browne et al. (J. Biol. Chem., 263:1599 [1988]) deleted the region between amino acids 57 and 81 and found the resulting variant to have a slower clearance from plasma. Collen et al. (Blood, 71:216 [1988]) deleted amino acids 6-86 (part of the finger and growth domains) and found that this mutant had a half-life in rabbits of 15 minutes as compared with 5 minutes for wild-type t-PA. Similarly, Kaylan et al. (J. Biol. Chem., 263:3971 [1988]) deleted amino acids 1-89 and found that the half-life of this mutant in mice was about fifteen minutes as compared to about two minutes for wild-type t-PA. Cambier et al. (I. Cardiovasc. Pharmacol., 11:468 [1988]) constructed a variant with the finger and growth factor domains deleted and the three asparagine glycosylation sites abolished. This variant was shown to have a longer half-life than wild-type t-PA when tested in dogs. Variants with only the growth factor domain or the finger domain deleted have also been demonstrated to have decreased clearance rates in rabbits, guinea pigs and rats (Higgins and Bennett, Ann. Rev. Pharmacol. Toxicol., 30:91 [1990] and references therein).
A variety of amino acid substitution t-PA variants have been evaluated for their ability to decrease the clearance rate or increase the half-life of t-PA. The variant R275E (where arginine at position 275 of native, mature t-PA was substituted with glutamic acid) has been shown to have a clearance rate of about two times slower than that of wild-type t-PA when tested in primates and rabbits (Hotchkiss et al., Thromb. Haemost., 58:491 [1987]). Substitutions in the region of amino acids 63-72 of mature, native t-PA, and especially at positions 67 and 68, have been reported to increase the plasma half-life of t-PA (see WO 89/12681, published Dec. 28, 1989).
Production of other substitution variants has focused on converting the glycosylation sites of t-PA to non-glycosylated sites. Hotchkiss et al. (Thromb. Haemost., 60:255 [1988]) selectively removed oligosaccharide from the t-PA molecule, and demonstrated that the removal of these residues decreased the rate of clearance of t-PA when tested in rabbits. Removal of the high mannose oligosaccharide at position 117 using the enzyme endo--N-acetylglucosaminidase H (Endo-H) resulted in a rate of clearance that was decreased about two fold. Oxidation of nearly all oligosaccharide residues using sodium periodate resulted in a rate of clearance nearly three fold lower than wild-type t-PA. These researchers also generated the t-PA variant N117Q (wherein asparagine at position 117 of native, mature t-PA was substituted with glutamine) to prevent glycosylation at position 117. The clearance rate of this variant was lower than wild-type t-PA. See also EP 238,304 published Sep. 23, 1987 and EP 227,462 published Jul. 1, 1987.
An additional approach to produce t-PA variants with extended circulatory half-life and slower clearance has been to add glycosylation sites to the molecule. As examples of this approach, positions 60, 64, 65, 66, 67, 78, 79, 80, 81, 82, and 103 have been substituted with appropriate amino acids to create molecules with glycosylation sites at or near some of these residues (see WO 89/11531, published Nov. 30, 1989 and U.S. Ser. No. 07/480,691, filed Feb. 15, 1990).
A general review of plasminogen activators and second-generation derivatives thereof can be found in Harris, Protein Engineering, 1: 449-458 (1987). Other reviews of t-PA variants include Pannekoek et al., Fibrinolysis, 2: 123-132 (1988), Ross et al., in Annual Reports in Medicinal Chemistry, Vol. 23, Chapter 12 (1988), and Higgins and Bennett, supra.
Accordingly, it is an object of this invention to provide a rapid and effective method for the systematic preparation of candidate binding substances.
It is another object of this invention to prepare candidate binding substances that are displayed on the surface of mammalian cells and that are conformationally stable.
It is still another object of this invention to prepare candidate binding substances comprising fusion proteins of a transmembrane anchor polypeptide and a heterologous protein where the heterologous protein is non-membranous and soluble, especially a secretory protein, and displayed on a cell, especially mammalian, where the fusion protein is encoded by DNA contained in a plasmid and the plasmid is transfected into a host cell.
It is a further object of this invention to provide a method for the preparation and selection of desired variant binding proteins employing a bivariant selection process in which the proteins are expressed on a mammalian cell surface and in which the proteins retain at least one binding property in common with, and one binding property different from the corresponding wild-type protein.
It is a still further object of this invention to provide novel t-PA mutants or variants having diminished binding affinity for receptors responsible for t-PA clearance.
These and other objects of this invention will be apparent from consideration of the invention as a whole.